Chromosomal evolution and evidence of a putative multiple sex chromosome system in the Amazonian northeastern pepper frog (<i>Leptodactylus vastus</i>, Lutz 1930)

The genus <i>Leptodactylus</i> is among the most studied anurans in cytogenetics. Despite exhibiting chromosomal conservatism with 2n = 22, microstructural studies (here defined as investigations of subchromosomal features such as DNA repetitive elements, chromatin organization, and sequence-level structural arrangements) have revealed significant karyotypic diversity within the genus, primarily associated with the distribution of repetitive sequences. Additionally, complex sex chromosome systems have been identified, underscoring the importance of further studies on these species. In this context, we performed a karyotypic characterization of <i>Leptodactylus vastus</i> from the Amazonian region. Using conventional cytogenetics, FISH with repetitive sequences, and phylogenetic analyses, we identified extensive chromosomal rearrangements, evidenced by the distribution of heterochromatin and repetitive sequences, including microsatellites, 18S rDNA, and U2 snDNA. Meiotic analyses in males revealed the presence of multivalent chromosomal chains involving eight chromosomes, a pattern consistent with a multiple sex chromosome system (X₁Y₁X₂Y₂X₃Y₃X₄Y₄). Concerning the clusters of repetitive sequences, we observed in specific chromosome pairs underscores the potential cytotaxonomic relevance of these markers. Phylogenetically, <i>Leptodactylus vastus</i> belongs to the <i>Leptodactylus pentadactylus</i> group, which includes other species known to possess multiple sex chromosome systems, although the absence of female data prevents definitive confirmation of this system in this species. Our results suggest that chromosomal rearrangements may have contributed to the genomic diversification of the species, supporting previous hypotheses on the role of structural variation in <i>Leptodactylus</i> species while highlighting the need for further comparative studies involving both sexes to fully clarify the nature of the multivalent chromosomal chains observed in <i>Leptodactylus vastus</i>.